Analysis in
maple.sgc.loc/home/bp2582/projects/eye-reg_rnaseq/github/SnailEyeReg_RNASeq/code/ExploreDataset.Rmd
This exploratory analysis aims to evaluate the quality of the data by
verifying that the relationships between samples make sense.
Comments: All samples, and specifically, replicates
within a sample-group show similar expression profiles which is
good.
Comments: Here we are looking for similarity between
the sample peeks. We want to see that all samples have similar peek
dynamic. We don’t want to see shifted peaks. In our data, the
distribution of counts in all samples is similar which is good. In the
1dpa samples, the higher counts seem less and the lower more compared to
all the other samples. Its not bad but lets keep an eye on that.
Comments: From https://support.illumina.com/bulletins/2017/04/considerations-for-rna-seq-read-length-and-coverage-.html.
Experiments looking for a more global view of gene expression, and some
information on alternative splicing, typically require 30–60 million
reads per sample. This range encompasses most published RNA-Seq
experiments for mRNA/whole transcriptome sequencing.
All of our samples have >30 million reads per sample so they are above the acceptable range.
We are calculating distances to evaluate similarity between samples.
Calculating distances must be done in normalized data. The data was
normalized with DESEQ transformation.
Comments:
- The replicates for each time point group nicely, except for the
timepoint 21 and 28 which are grouping together.
- It makes sense that 1) 1 dpa is very isolated from the rest, 2) 3 and
6 dpa group together, 3) 21 and 28 group together, 4) Intact groups with
the more advances timepoints (9 dpa +). The only relationship that
caughts my attention is that, according to this plot, 9 and 15 dpa are
more similar between them than with 12 dpa. Nevertheless, this looks
good to me.
Comments: Dim1 and Dim2 separate nicely the timepoints. There is a clear division between the 1, 3, 6, 9 dpa and intact groups. There is a messy cloud that includes 12, 15, 21 and 28 dpa. Dim 3 and Dim4 are not informative.
Comments: In the PCA plot, Dim1 and Dim2 also separate
nicely the timepoints. There is a clear division between the 1, 3, 6, 12
dpa and intact groups. There is one cloud that groups 9 with 15 and 21
with 28. This agrees with the relationships shown in the euclidian
distances plot. Dim 3 and Dim4 are not informative.
Comments:/ - Separates nicely the intact eye/ - 3 and 6
dpa form their one groups but also we see a high correlation between
both of them/ - And then we have the 12, 15, 21, 28 dpa cloud
Comments:/ - This shows higher correlation between
replicates of the same group, altough we see In a same “cloud” 28 and 21
dpa, and 9 and 15 dpa.
Comments: Using 100 genes the Spearman correlation
shows higher correlation between replicates of same sample. Using
Pearson 9, 12, 15, 21 and 28 are higly correlated.